The brain renin-angiotensin system has long been recognized as an important determinant of arterial pressure regulation. It is now well documented that Ang-ll is synthesized in the brain and has been detected in nuclei regulating the cardiovascular (CV) system and in neuronal fibers connecting some of these pathways. We recently localized both renin- and AGT-expressing neurons in nuclei extensively interconnected by angiotensinergic fibers, and have identified two forms of renin in the brain: a) the classical secreted renin termed sREN, and b) a unique form of constitutively active renin termed icREN that lacks a signal (secretory) peptide and the first third of the prosegment and thus remains intracellular. We provide preliminary evidence supporting the concept that Ang-ll synthesized by the intracellular renin pathway plays a role in CV regulation. We hypothesize that neuronal production of this brain-specific isoform of intracellular active renin completes the biosynthetic pathway for the intracellular generation of Ang-ll in neurons. In the brain therefore, Ang-ll is formed by two mechanisms: 1) a classical extracellular pathway utilizing AGT secreted mainly from astrocytes and renin secreted from neurons, and 2) a novel intracellular pathway in neurons co-expressing both AGT and intracellular active renin. We further hypothesize that the intracellular pathway for Ang-ll synthesis initiated by icREN plays an important role in CV regulation. We will take advantage of the unique expertise of investigators in this Program, to test for the first time the physiological significance of intracellular renin and Ang-ll in the brain and relate it to Ang-ll-dependent mechanisms of CV control. We will accomplish this through studies proposed in two Specific Aims: 1. Define the location and properties of icREN-expressing neurons by testing the hypothesis that they co-express AGT, ACE and Ang- ll, have cell bodies localized in nuclei controlling cardiovascular function, and have axons which project within known angiotensinergic neural pathways controlling cardiovascular function. We will accomplish this by developing and characterizing novel transgenic mouse models expressing easily detectable reporter genes under the control of the brain-specific icREN promoter or the classical sREN promoter. 2. Define the functional relevance of icREN by testing the hypothesis that this pathway is a critical regulator of CV function. We will accomplish this by examining blood pressure regulation and other Ang-ll-dependent cardiovascular end points in mice either over-expressing icREN or sREN, and in knockout mice lacking icREN but preserving sREN. Blood pressure will be examined under baseline conditions, in response to restraint stress, and in experimental hypertension. We anticipate that these studies will provide fundamental evidence conceptually advancing our understanding of the mechanisms controlling the production Ang-ll in the brain under normal conditions and in hypertension.